PMID: 11337045May 5, 2001Paper

Development of a strand-specific RT-PCR based assay to detect the replicative form of hepatitis C virus RNA

Journal of Virological Methods
J K CraggsA M Grabowska

Abstract

The recent development of tagged RT-PCR and rTth RT-PCR has greatly improved strand-specific detection of hepatitis C virus (HCV) RNA but these assays are still prone to some false detection of the incorrect strand of RNA. In this study we aimed to address additional factors which contribute towards false detection of HCV RNA. Firstly the benefits of both tagged primers and the thermostable reverse transcriptase rTth during cDNA synthesis were combined and it was found that strand specificity was greatly improved without compromising sensitivity. The reliability of the assay was then optimised by addressing the following issues: control synthetic transcripts should be free of contaminating plasmid DNA, residual RT activity should be minimised in the presence of PCR primers and cDNA should be free of unincorporated tagged RT primer prior to PCR amplification. The alterations made to the assay eliminated completely false detection of the incorrect strand of RNA in the control assay whilst the correct strand was consistently detected at a cDNA dilution of 10(-3)-10(-4). Negative strand was not detected in RNA isolated from serum but was detected, at a ten-fold lower level than positive strand, in RNA isolated from liver tissue.

References

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Citations

Jun 15, 2010·Archives of Virology·So Young JangChan Hee Lee
Jan 13, 2004·Journal of Virological Methods·Florence Komurian-PradelPatrice André
May 27, 2008·Genome Research·Qian-Ru LiMarc Vidal
Jul 21, 2011·Conservation Biology : the Journal of the Society for Conservation Biology·Ivan MeeusGuy Smagghe
Apr 15, 2003·Journal of Virology·Baohua GuRobert T Sarisky
Dec 28, 2010·Journal of Virological Methods·Eiryo KawakamiYoshihiro Kawaoka
Aug 4, 2009·Journal of Virological Methods·Steven M VallesJuan Briano
Jun 15, 2007·Journal of Virological Methods·Jacquelyn Horsington, Zhidong Zhang
Jun 25, 2014·Scientific Reports·B Uma ReddySaumitra Das
Oct 31, 2009·Liver Transplantation : Official Publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society·Michael G Hughes, Hugo R Rosen
Jul 24, 2008·Journal of Medical Virology·Javier BartoloméVicente Carreño
Jan 15, 2009·The Journal of General Virology·Sebastian GisderElke Genersch
Nov 27, 2009·The Journal of General Virology·A TuiskunenS Plumet
Jul 15, 2015·Archives of Virology·Steven M Valles, Sanford D Porter
Aug 24, 2013·Journal of Virological Methods·Stephanie M LimByron E E Martina
May 15, 2013·Journal of Invertebrate Pathology·Sanford D PorterDavid H Oi
Sep 24, 2011·Journal of Invertebrate Pathology·Omaththage P PereraRandall G Luttrell
Dec 21, 2014·Virus Research·Chang-Hee KweonSeung-Won Kang

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